Modified beta-lactamases and methods and uses related thereto

ABSTRACT

The present invention relates to pharmaceuticals and modified beta-lactamases. Specifically, the invention relates to novel recombinant beta-lactamases and pharmaceutical compositions comprising the beta-lactamases. 
     Also, the present invention relates to methods for modifying a beta-lactamase, producing the beta-lactamase and treating or preventing beta-lactam antibiotic induced adverse effects. Furthermore, the present invention relates to the beta-lactamase for use as a medicament and to the use of the beta-lactamase in the manufacture of a medicament for treating or preventing beta-lactam antibiotics induced adverse effects. 
     Still further, the invention relates to a polynucleotide and a host cell comprising the polynucleotide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/138,767, filed Apr. 26, 2016, which is a continuation of U.S. patentapplication Ser. No. 15/054,292, filed Feb. 26, 2016 (now U.S. Pat. No.9,587,234), which is a continuation of U.S. patent application Ser. No.14/676,559, filed Apr. 1, 2015 (now U.S. Pat. No. 9,301,996), which is acontinuation of U.S. patent application Ser. No. 14/517,539, filed Oct.17, 2014 (now U.S. Pat. No. 9,301,995), which is a continuation of U.S.patent application Ser. No. 14/047,882, filed Oct. 7, 2013 (now U.S.Pat. No. 8,894,994), which is a continuation of U.S. patent applicationSer. No. 13/699,434, filed Nov. 21, 2012 (now U.S. Pat. No. 9,034,602),which is a U.S. National Stage Application of International ApplicationNo. PCT/FI2011/050450, filed May 17, 2011, which claims priority fromFinnish Patent Application No. 20105572, filed May 24, 2010, the entirecontents of all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceuticals and modifiedbeta-lactamases. Specifically, the invention relates to novelrecombinant beta-lactamases and pharmaceutical compositions comprisingthe beta-lactamases.

Also, the present invention relates to methods for modifying abeta-lactamases, producing the beta-lactamase and treating or preventingbeta-lactam antibiotic induced adverse effects. Furthermore, the presentinvention relates to the beta-lactamase for use as a medicament and tothe use of the beta-lactamase in the manufacture of a medicament fortreating or preventing beta-lactam antibiotics induced adverse effects.

Still further, the invention relates to a polynucleotide and a host cellcomprising the polynucleotide.

BACKGROUND OF THE INVENTION

Beta-lactam antibiotics are characterized by a beta-lactam ring in theirmolecular structure. The integrity of the beta-lactam ring is essentialfor the biological activity, which results in the inactivation of a setof transpeptidases that catalyze the final cross-linking reactions ofpeptidoglycan synthesis. Members of the beta-lactam antibiotics familycomprise penicillins, cephalosporins, clavams (or oxapenams),cephamycins and carbapenems.

Beta-lactamases are bacterial defensive enzymes that hydrolyzebeta-lactam antibiotics. The production of beta-lactamases is apredominant mechanism to confer beta-lactam resistance in Gram-negativebacteria. Beta-lactamases catalyse very efficiently irreversiblehydrolysis of the amide bond of the beta-lactam ring resulting inbiologically inactive product(s).

Because of the diversity of enzymatic characteristics of differentbeta-lactamase types, several classification systems have been proposedfor their categorising. The classifications are based on two majorapproaches, which are functional and molecular classifications.

The functional classification scheme of beta-lactamases proposed by Bushet al. (1995, Antimicrob. Agents Chemother. 39: 1211-1233) defines fourbeta-lactamase groups, which are based on their substrate and inhibitorprofiles. Group 1 consists of cephalosporinases that are not wellinhibited by clavulanic acid. Group 2 consists of penicillinases,cephalosporinases and broad-spectrum beta-lactamases that are generallyinhibited by active site-directed beta-lactamase inhibitors. Group 3consists of melallo-beta-lactamases that hydrolyze penicillins,cephalosporins and carbapenems, and that are poorly inhibited by almostall beta-lactam-containing molecules. Group 4 consists of penicillinasesthat are not well inhibited by clavulanic acid. Sub-groups have alsobeen defined according to rates of hydrolysis of carbenicillin orcloxacillin (oxacillin) by group 2 penicillinases.

The most widely used classification is Ambler classification whichdivides beta-lactamases into four classes (A, B, C, D) and is based ontheir amino-acid sequences (Ambler 1980, Philos Trans R Soc Lond B BiolSci. 289: 321-331). Classes A, C, and D gather evolutionarily distinctgroups of serine beta-lactamase enzymes, and class B the zinc-dependent(“EDTA-inhibited”) beta-lactamase enzymes (Ambler R. P. et al., 1991,Biochem J. 276: 269-270). Classes A, C, and D belong to serinebeta-lactamases, in which the hydrolysis of the beta-lactam is mediatedby serine in an active site. Serine beta-lactamases are related to DDpeptidases (D-alanyl-D-alanine carboxypepti-dase), the target enzyme ofbeta-lactams. The mechanism by which serine beta-lactamases hydrolyzebeta-lactam antibiotics is believed to follow a three-step pathwayincluding a non-covalent Henri-Michaelis complex, a covalent acyl-enzymeintermediate and deacylation (Matagne et al., 1998, Biochem J330:581-594 Acylation mechanism is considered to be a common mechanismfor all serine beta-lactamase groups whereas, on the basis oftheoretical calculations, the substrate deacylation mechanisms of serinebeta-lactamase of classes A, C and D appear to differ from each other.Deacylation mechanisms have both common and group specific elementaryprocesses (Hata M et al., 2006, Biol Pharm Bull. 29: 2151-2159).

Bacillus spp. serine beta-lactamases and TEM-1, SHV-1 and CTX-M familieshave primarily been classified as class A beta-lactamases and aspenicillinases that possess good capability to hydrolyze e.g. penicillinand ampicillin. The class A beta-lactamases were first identified inpenicillin resistant St. aureus in the 1940s. A plasmid-borne penicillinresistance gene, TEM-1, was discovered in E. coli 20 years later. Lateron, serine beta-lactamases were also shown to evolve the ability tohydrolyze most cephalosporins and further specialize at hydrolysing aspecific subset of cephalosporins. Most of these extended-spectrumbeta-lactamases (ESBL) are derivates of TEM-1, TEM-2 or SHV-1 enzymes.Recently there are increasing numbers of reports that describe the vastemergence of CTX-M enzymes, a new group of class A ESBLs. Nowadays,CTX-M enzymes are the most frequently observed ESBLs and aresub-classified into five major families. CTX-M enzymes have a widesubstrate range including penicillin and the first, second and thirdgeneration cephalosporins (Bonnet, R. 2004. Antimicrob Agents Chemother.48:1-14).

While the sequence similarity between the class A beta-lactamases (TEM,SHV, CTX-M, Bacillus spp. beta-lactamases) is moderate, the crystalstructures of all serine beta-lactamases show a particularly highsimilarity (Matagne et al., 1998, Biochem J 330:581-598: Tranier S. etal., 2000, J Biol Chem, 275: 28075-28082; Santillana E. et al., 2007,Proc Natl Acad Sci. USA, 104; 5354-5359). The enzymes are composed oftwo domains. One domain consists of a five-stranded beta sheet packedagainst three alpha helices whilst the second domain, an alpha domain,is composed of eight alpha helices. The active site pocket is part ofthe interface between these two domains and is limited by the omegaloop. The omega loop is a conserved structural element of all class Abeta-lactamases and is essentially involved in catalytic reaction (FIG.1).

Several conserved peptide sequences (elements) related to catalysis orrecognition of the substrate have been identified in class Abeta-lactamases. The first conserved element 70-Ser-X-X-Lys-73 (Amblerclassification) includes the active serine residue at location 70 inalpha helix₂ and lysine at position 73. The second conserved element isa SXN loop in an alpha domain (at positions between 130 and 132according to Ambler classification), where it forms one side of acatalytic cavity. The third conserved element (at positions between 234and 236 according to Ambler classification) is on the innermost strandof the beta-sheet₃ and forms the other side of the catalytic cavity. Thethird conserved element is usually KTG. However, in some exceptionalcases, lysine (K) can be replaced by histidine (H) or arginine (R), andin several beta-lactamases, threonine (T) can be substituted by serine(S) (Matagne et al., 1998. Biochem 330:581-598).

Beta-lactamase mediated resistance to beta-lactams is widely spreadamong pathogen and commensal microbiota, because of abundant use ofbeta-lactams in recent decades. Indeed, antibiotic resistance is awell-known clinical problem in human and veterinary medicine, andhundreds of different beta-lactamases derived from Gram-positive andGram-negative bacteria have been purified and characterized in thescientific literature. Because the use of antimicrobials has not reducedand furthermore, antimicrobial resistance has become part of theeveryday life, new approaches are invariably and urgently required forsolving these medical problems.

The intestinal microbiota of humans is a complex bacterial communitythat plays an important role in human health, for example, bystimulating, the immune response system, aiding in digestion of food andpreventing the overgrowth of potential pathogen bacteria. Antimicrobialagents e.g. beta-lactams are known to have effect on normal microbiota.The efficacy of antimicrobial agents to promote changes of normalintestinal microbiota is associated with several factors including drugdosage, route of administration and pharmacokinetics/dynamics andproperties of antibiotics (Sullivan Å. et. al., 2001, Lancet 11101-114).Even though the intestinal microbiota have a tendency to revert tonormal after completion of antibiotic treatment, long term persistenceof selected resistant commensal bacteria has been reported (Sjölund M.et al., 2003, Ann Intern Med. 139:483-487). Such persistence and theexchange of antibiotic, resistance genes make the commensal microbiota aputative reservoir of antibiotic resistance genes.

Certain parentally administered beta-lactams like ampicillin,ceftriaxone, cefoperazone, and piperacillin are in part eliminated viabiliary excretion into the proximal part of the small intestine(duodenum). Residual unabsorbed beta-lactams in the intestinal tract maycause an undesirable effect on the ecological balance of normalintestinal microbiota resulting in antibiotic-associated diarrhea,overgrowth of pathogenic bacteria such as vancomycin resistantenterococci (VRE), extended-beta-lactamase producing Gram-negativebacilli (ESBL), Clostridium difficile, and fungi, and selection ofantibiotic-resistance strains among both normal intestinal microbiotaand potential pathogen bacteria.

The therapeutic purpose of beta-lactamases is inactivating unabsorbedantibiotics in the gastrointestinal tract (GIT), thereby maintaining anormal intestinal microbiota and preventing its overgrowth withpotentially pathogenic micro-organisms (WO 93/13795).

There are at least three requirements for beta-lactamase drug products,which are suitable for GIT targeted therapy. The first requirement is topreserve enzymatic activity under conditions prevailing in the GIT.Resistance against proteolytic breakdown by various proteases secretedfrom various glands into the GIT is a quintessential precondition forthe feasibility of beta-lactamase therapy. Another importantconsideration is the range of pH values prevailing in the differentcompartments of the small intestine. These pH values usually varybetween 5 (duodenum) and 7.5 (ileum). Hence, in order to qualify ascandidates for the intended therapeutic purpose, a beta-lactamase needsto exhibit high enzymatic activity over the pH range 5-7.5.

The second requirement of a beta-lactamase or a product thereof is tohydrolyze beta-lactam efficiently. The concentration of a beta-lactamantibiotic in small intestinal chyme during an antibiotic treatmentepisode is mostly related to the elimination of the particularbeta-lactam via biliary excretion. A suitable beta-lactamase needs tohave kinetic parameters that enable it to effectively hydrolyze lowerGIT beta-lactam concentrations below levels causing alterations inintestinal microbiota. The ideal set of kinetic parameters consists of anumerical low value for the Michaelis constant K_(M), combined with anumerically high value for the maximum reaction rate V_(max). A highV_(max) value is required in order to provide a sufficient degree ofbreakdown capacity, while a low K_(M) value is needed to ensurebeta-lactam degrading activity at low substrate concentrations.

The third requirement of a beta-lactamase or a product thereof is totolerate the conditions, such as relatively high temperatures, in themanufacturing of pharmaceutical compositions. Moreover, in theproduction process, the mixing dispersion of aqueous excipients and drugsubstance requires a high degree of solubility at suitable pH.

An enzymatic therapy, named Ipsat P1A, is being developed for theprevention of the adverse effects of β-lactam antibiotics inside thegut. Ipsat P1A delivery system has been designed to inactivateparenterally given penicillin group beta-lactams (e.g. penicillin,amoxicillin ampicillin and piperacillin) with or without beta-lactamaseinhibitors (e.g. tazobactam, sulbactam, clavulanic acid) excreted viabiliary system (WO 2008065247; Tarkkanen, A. M. et al., 2009, AntimicrobAgents Chemother. 53:2455-2462). The P1A enzyme is a recombinant form ofBacillus licheniformis 749/C small exo beta-lactamase (WO 2008065247)which belongs to class A and is grouped to subgroup 2a in functionalclassification. B. licheniformis beta-lactamase and its P1A derivate areconsidered as penicillinases which have high hydrolytic capacity todegrade e.g. penicillin, ampicillin, amoxicillin or piperacillin(Table 1) and they are generally inhibited by active site-directedbeta-lactamase inhibitors such as clavulanic acid, sulbactam ortazobactam (Bush K. et al., 1995, Antimicrob Agents Chemother 39:1211-1233).

However, the P1A enzyme has only a very limited capacity to inactivatebeta-lactam antibiotics that belong to the cephalosporin or thecarbapenem group. Because the employed beta-lactamases possess pooractivity to cephalosporins, they can not be applied in conjunction withparenteral cephalosporin therapy for inactivation of unabsorbedbeta-lactam in the small intestinal tract.

Therefore, new beta-lactamases or derivates of P1A with extendedsubstrate profile, for example as seen in metallo-beta-lactamases, areindispensable.

The present invention provides novel genetically tailored derivates ofP1A beta-lactamase and furthermore, novel methods for modifying andproducing beta-lactamases.

BRIEF DESCRIPTION OF THE INVENTION

The new recombinant derivates of P1A beta-lactamase of the inventionfulfill the above-mentioned three requirements of suitablebeta-lactamases (i.e. have abilities to preserve enzymatic activity,hydrolyze beta-lactams efficiently and tolerate conditions in themanufacturing of the pharmaceutical compositions) and furthermore, haveextended substrate profiles. The beta-lactamases of the invention mayalso be used in conjunction with parenteral cephalosporin therapy forinactivating binary eliminated beta-lactam in the small intestinaltract.

The present invention highlights the preliminary and preclinical studiesof a new Ipsat P3A pharmaceutical protein (a D276N substituted derivateof P1A) and presents a single drug substance dose.

The present invention enables rapid and efficient methods for modifyingbeta-lactamases and for producing them. Furthermore, by the presentinvention more effective and specific treatments become available.

The enzymes of the invention are suitable for large scale manufacturingfor a drug substance for treating or preventing adverse effects inducedby various groups of beta-lactam antibiotics.

The object of the present invention is to provide novel beta-lactamases,especially beta-lactamases of B. licheniformis, and to provide products,methods and uses related to the beta-lactamases. Tools for furtherdevelopments in pharmaceutical industries are also presented by theinvention.

The present invention relates to a beta-lactamase comprising an aminoacid sequence having at least 60% sequence identity with SEQ ID NO: 1and having a hydrophilic amino acid residue at a position of SEQ ID NO:1 corresponding to position 276 according to Ambler classification, or avariant or fragment thereof.

The invention also relates to a pharmaceutical composition comprisingbeta-lactamase of the invention.

The invention also relates to a method of modifying a beta-lactamasecomprising an amino acid sequence having at least 60% sequence identitywith SEQ ID NO: 1, wherein an amino acid of the beta-lactamase at aposition of SEQ ID NO: 1 corresponding to position 276 according toAmbler classification is replaced with a hydrophilic amino acid.

Furthermore, the invention relates to a method of producing thebeta-lactamase, wherein the method comprises the following steps:

i) providing a gene encoding the beta-lactamase of the invention;

ii) transforming a host cell with the gene;

iii) obtaining a host cell that produces the beta-lactamase;

iv) recovering the beta-lactamase.

Furthermore, the invention relates to a method of treating or preventingbeta-lactam antibiotic induced adverse effects in the gastro-intestinaltract by administering beta-lactamase of the invention simultaneously orsequentially with a beta-lactam antibiotic to a subject.

Still further, the present invention relates to the beta-lactamase foruse as a medicament.

Still further, the present invention relates to a use of thebeta-lactamase in the manufacture of a medicament for treating orpreventing beta-lactam antibiotics induced adverse effects in thegastro-intestinal tract.

Still further, the invention relates to a polynucleotide, whichcomprises a sequence of any one of SEQ ID NO:s 2 or 4 or a degeneratethereof, or it encodes the beta-lactamase of the invention. Theinvention also relates to a host cell comprising the polynucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the 3D structure of beta-lactamase of Bacilluslicheniformis beta-lactamase (small exo form of PenP). The conservedamino acid residues and the side chains residues of R-244 and D-278 aremarked. The diagram was generated by using MolSof-Browser programme.

FIG. 2 shows the nucleotide and deduced amino acid sequences of D276Nbeta-lactamase gene of Bacillus licheniformis (P1A derivate). The aminoacid sequence corresponds to sequence SEQ ID NO: 3, wherein Xaa isasparagine (Asn). The nucleotide sequence corresponds to sequence SEQ IDNO: 4, wherein the nucleotide triplet nnn is aat. The open reading frameencodes a 299 amino acid polypeptide possessing a 31 amino acid longsignal sequence (underlined) of the amyQ gene derived from the pKTH141secretion vector (WO 2008/065247). The predicted signal peptidasecleavage site is after alanine (A) at position −1. The HindIII cloningsite that encodes an NH₂-QAS extension is expressed as bold. The matureD276N mutant enzyme starts from glutamine (Q) at a position of +1. Thus,the mature D276N mutant beta-lactamase comprises 268 amino acid residuesincluding the NH₂-QAS extension encoded by HindIII. A single amino acidsubstitution of aspartic acid (D) to asparagine (N) is located at theposition 280 (expressed as a bold character) corresponding to theposition of 276 in the Ambler classification system and corresponding toamino acid position 249 in sequence SEQ ID NO: 3.

The NH₂-terminal sequence of purified D276N mutant enzyme was determinedby automated Edman degradation in a protein sequencer. Analysisdemonstrated that the D276N mutant enzyme lacks NH₂-QASKT-pentapeptideat its deduced amino terminus in a manner similar to that of its parentP1A enzyme (WO 2008/065247). The major fraction of the purified D276Nmutant enzyme, which has been utilized in examples 4 and 6 of thisapplication, initiates from glutamic acid at position +6 and is composedof 263 amino acid residues with a molecular mass of 29 272.

FIG. 3 shows the nucleotide and deduced amino acid sequences of D276Rsubstituted beta-lactamase gene of P1A derived from BacillusIicheniformis. The amino acid sequence corresponds to sequence SEQ IDNO: 3, wherein Xaa is arginine (Arg). The nucleotide sequencecorresponds to sequence SEQ ID NO: 4, wherein the nucleotide triplet nnnis cgc.

FIG. 4 shows the effect of orally administered enteric coated D276Nsubstituted beta-lactamase (P3A) pellets on the concentrations ofceftriaxone in jejunal chyme of beagle dogs (n=5) after intravenousadministration of ceftriaxone (30 mg of ceftriaxone per kg of bodyweight) (closed squares). Beta-lactamase pellets were received 10minutes prior to ceftriaxone injection. Closed diamonds represent jejunaceftriaxone concentrations achieved after a single dose of ceftriaxone(i.v.) without beta-lactamase treatment.

DETAILED DESCRIPTION OF THE INVENTION

Beta-lactamases have been used in inactivating unabsorbed beta-lactamsin the gastrointestinal tract in order to prevent the beta-lactaminduced adverse effects including alterations in intestinal normalmicrobiota and the overgrowth of beta-lactam resistant bacteria (WO9313795, WO 2008065247, WO 2007147945. The present invention nowprovides a modified beta-lactamase of Bacillus licheniformis, whichshows a surprising altered substrate profile.

As used herein, a beta-lactamase refers to an enzyme, which hydrolyzesbeta-lactams. Hydrolysis of the amide bond of the beta-lactam ring makesthe antimicrobial agents biologically inactive. As used herein, class Abeta-lactamases (Ambler classification) refer to serine beta-lactamases,in which hydrolysis of beta-lactam is mediated by serine in the activesite, usually at amino acid position 70 in the alpha helix₂. Class Abeta-lactamases include but are not limited to Len-1, SHV-1, TEM-1,PSE-3/PSE-3, ROB-1, Bacillus cereus such as 5/B type 1, 569/H type 1 and569/H type 3, Bacillus anthrasis sp, Bacillus licheniformis such asPenP, Bacillus weihenstephanensis, Bacillus ciausii, Staphylococcusaureus, PC1, Sme-1, NmcA, IMI-, PER-, VEB-, GES-, KPC-, CME- and CTX-Mtypes beta-lactamases.

Sequence Identity of Peptides and Polynucleotides

The amino acid sequences of the mutant beta-lactamase of the presentinvention (D276X, P1A derivate) are set forth as SEQ ID NO: 1 and SEQ IDNO: 3. The corresponding nucleotide sequences are set forth as SEQ IDNO: 2 and SEQ ID NO: 4. SEQ ID NO: 1 sets forth the amino acid sequencetaking part in the formation of secondary structure of thebeta-lactamase. SEQ ID NO: 3 sets forth the full length amino acidsequence of the protein, including the 31 amino acids long signalsequence.

A beta-lactamase of the invention may comprise an amino acid sequencehaving at least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 99.5, 99.8, 99.9 or 100% identity with SEQ ID NO: 1 or 3.

According to a specific embodiment of the invention, the peptide has atleast 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 37, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 99.5, 99.8, 99.9 or 100% identity with SEQ ID NO: 1 or 3.

In one preferred embodiment of the invention, the beta-lactamase of theinvention comprises an amino acid sequence having at least 60% sequenceidentity with SEQ ID NO: 1. In another preferred embodiment of theinvention the beta-lactamase has at least 60% sequence identity with SEQID NO: 1 or 3.

In one embodiment of the invention the beta-lactamase comprising anamino acid sequence having any above-mentioned sequence identity withSEQ ID NO: 1, has a hydrophilic amino acid selected from a groupconsisting of arginine (R), histidine (H), lysine (K), asparagine (N),glutamine (Q), serine (S) and threonine (T) at a position of SEQ ID NO:1 corresponding to position 276 according to Ambler classification.

In a preferred embodiment of the invention the peptide has the sequenceshown in SEQ ID NO: 1 or 3. In one embodiment of the invention, thebeta-lactamase has the sequence as shown in SEQ ID NO: 1 or 3, wherein ahydrophilic amino acid residue at a position corresponding to position276 according to Ambler classification (marked as Xaa in SEQ ID NO: 1 or3) is an arginine (R, Arg). In another embodiment of the invention, thebeta-lactamase has the sequence as shown in SEQ ID NO: 1 or 3, wherein ahydrophilic amino acid residue at a position corresponding to position276 according to Ambler classification (marked as Xaa in SEQ ID NO: 1 or3) is an asparagine (N, Asn).

Identity of any sequence with the sequence of this invention refers tothe identity with the entire sequence of the present invention. Sequenceidentity may be determined by any conventional bioinformatic method, forexample by using BLAST (Basic Local Alignment Search Tools) or FASTA(FAST-All).

The present invention also relates to any variants or fragments of thenovel beta-lactamases. As used herein, a fragment or variant of thebeta-lactamase refers to any part or variant, which has a biologicalfunction i.e. is enzymatically active. A variant refers to a peptidehaving small alterations in the peptide sequence, e.g. mutations, smalldeletions or insertions. The fragments and variants should include thehydrophilic, amino add at a position corresponding to position 276according to Ambler classification. The hydrophilic amino acid istypically other than aspartic acid (D).

There are various short forms of the beta-lactamase, which areobtainable from SEQ ID NO: 3 and which are secreted outside the cell.These are called exoforms. The exoforms are the result of hydrolyticactivity of proteases in the cell wall or culture medium.

D276X, D276N, D276R, mutant form, P1A derivate or P3A, as used hereinencompasses any beta-lactamase active fragment and/or variant of the SEQID NO: 3 or variant comprising the explicitly given amino acid sequence(SEQ ID NO: 1). Especially, the beta-lactamase of the invention is anNH₂-truncated form, which means that it has been truncated at theaminoterminus. In addition to the NH₂-truncation, it may comprise one ormore further amino acid deletions, substitutions and/or insertions, aslong as it has beta-lactamase activity. Said modifications may be eithernaturally occurring variations or mutants, or artificial modificationsintroduced e.g. by gene technology.

Differently aminoterminally truncated exoforms have been found in thegrowth medium of B. lieheniformis. Such exoforms are also encompassedherein. Matagne et al. have described various extents ofmicroheterogeneity in extracellular forms produced by the natural hostB. ficheniformis 749/C (Matagne A. et al., 1991. Biochem J.273:503-510). The following five different secreted exoforms withdifferent N-terminal amino acid residues were identified:

SQPAEKNEKTEMKDD...KALNMNGK        EKTEMKDD...KALNMNGK        KTEMKDD...KALNMNGK           EMKDD...KALNMNGK           MKDD...KALNMNGK

initial amino acid residues are presented in bold. The C-terminal aminoacid residues are indicated to the right. The exoform starting fromserine (S) is called the “large secreted form” of B. licheniformisbeta-lactamase, and the one starting from lysine (K) is called the“small secreted form”.

The first alpha helix (α₁-helix) starts from aspartatic acid (D)(presented in italics) and the end of the last alpha helix (α₁₁-helix)ends at asparagine (N) (presented in italics). According to oneembodiment of the invention the beta-lactamase comprises at least theamino acids 1-258 of SEQ ID NO: 1 or amino acids 7-264 of SEQ ID NO: 3,which take part in the secondary structure of the protein (Knox J. R. etal., 1991. J. Mol Biol. 220: 435-455). According to another embodimentof the invention one or more of said amino acids 1-258 of SEQ ID NO: 1or amino acids 7-264 of SEQ ID NO: 3 have been deleted or replaced.

According to still another embodiment of the invention the aminoterminal of the beta-lactamase begins with NH₂-KTEMKDD (amino acids 4-10of SEQ ID NO: 3). This so-called ES-betaL exoform may further lack up to21 contiguous residues as described by Gebhard et al. (Gebhard L. G. etal., 2006, J. Mol. Biol. 21:358(1)280-288). According to anotherembodiment of the invention the amino terminal begins with glutamic acid(E) of SEQ ID NO: 3, and especially it begins with NH₂-EMKDD (aminoacids 6-10 of SEQ ID NO: 3), or alternatively it begins with NH₂-MKDD(amino acids 7-10 of SEQ ID NO: 3 or amino acids 1-4 of SEQ ID NO: 1).

The variable region in the amino terminal sequence of the beta-lactamasehas no rigid structure which accounts for the constancy of enzymaticparameters of various beta lactamase forms.

The four last amino acids at the carboxylic end of the beta-lactamase,MNGK-COOH (amino acids 265-268 of SEQ ID NO: 3), are not part of thesecondary structure, and may therefore also be deleted without loosingactivity. In another embodiment up to nine C-terminal amino acids may bedeleted. C-truncated forms of the protein have been described by Santoset al. (Santos J. et al., 2004. Biochemistry 43:1715-1723).

All the different forms of the beta-lactamase set forth above areencompassed by the present invention, together with other forms of theprotein having beta-lactamase activity.

A polynucleotide of the invention may comprise or have a sequence of anyone of SEQ ID NO: 2 or 4 or a degenerate thereof. A polynucleotide thatis a degenerate of a sequence shown in any one of SEQ ID NO:s 2 or 4refers to a polynucleotide that has one or more different nucleotidescompared to SEQ ID NO:s 2 or 4 but encodes for the same amino acid.Preferably, the nucleotide triplet nnn of SEQ ID NO: 2 or 4 encodes ahydrophilic amino acid, most preferably N or R. A “polynucleotide” asused herein is a sequence of nucleotides such as a DNA or RNA sequence,and may be a single or double stranded polynucleic acid. The termpolynucleotide encompasses genomic DNA, cDNA and mRNA.

According to a specific embodiment of the invention, the polynucleotidehas at least 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 99.5, 99.8 or 99.9% identity to any one of the nucleotidesequences of SEQ ID NO: 2 or 4, or fragments thereof.

In one specific embodiment of the invention the polynucleotide has asequence shown in any one of the sequences SEQ ID NO: 2 or 4.

Amino Acids at Position 276 (Ambler) of Class A Beta-lactamases

Asparagine (Asn, N) at amino acid position 276 is present in a widevariety of class A beta-lactamases. The function of Asn276 has beenintensively studied in TEM and SHV beta-lactamases, in which Asn276forms hydrogen bonds with the guanidium group of arginine (Arg, R) 244and thus, limits the mobility of the Arg244 side chain.

Substitutions of asparagine (Asn, N) in TEM or SHV enzymes have beenrecognised as one major contributor to resistance to serinebeta-lactamase inhibitors such as clavulanic adic sulbactam ortazobactam. N276D (Asp) substitution variants of TEM-1 beta-lactamaseare present in inhibitor resistant beta-lactamases (IRT enzymes such asTEM-35 and TEM-36). An N276D variant is more resistant to clavulanicacid and tazobactam than the wild type TEM-1 enzyme, but concomitantlythe catalytic efficiencies (kcat/Km) of N27513 variant for variouspenicillins are less than 50% of those in the TEM-1 wild type enzyme.Catalytic efficacies of the N276D variant to cephalosporins are reducedcompared to those of the wild type TEM-1 (Saves I et al., 1995, J BiolChem. 270:18240-18245).

Similarly to TEM-1, N276D substitution in SHV-1 or SHV-5 beta-lactamaseenhances the resistance to serine beta-lactamase inhibitors but reducestheir hydrolytic efficiencies to most beta-lactams (Giakkoupi P. et al.,1999, J Antimicrobial Chemother, 43: 23-29). Furthermore, N276Dsubstitution in SHV-1 or SHV-5 enzymes moderately improves their abilityto degrade “fourth generation” cephalosporins cefpirome and cefepime.

In SHV type beta-lactamase OHIO-1, an N276G (Gly) mutant has shown to behighly resistant to clavulanic acid, whereas a TEM-1 derived N276Gmutant possesses only moderate resistance to clavulanic acid (Bonomo R Aet al., 1995, Biochim Biophys Acta. 1247:121-125).

In the family of CTX-M enzymes, arginine (Arg, R) is typically presentat position 276 (Bonnet R. 2004, Antimicrob Agents Chemother, 48: 1-14)and mutations of Arg276 affect the extension of enzyme activity.Relative hydrolysis rates of CTX-M enzymes against cefotaxime aremoderately reduced by substitution of Arg276. Furthermore, Arg276Trp,Arg276Cys, Arg276Ser and Arg276Gly CTX-M mutant enzymes do not affectthe level of beta-lactamase inhibitor resistance (Bonnet R., 2004,Antimicrob Agents Chemother. 48: 1-14; Perez-Llarena F. J. et al., 2008J Antimicrobiol Chemother, 61 792-797).

TABLE 1 Amino acid residues located at 276 position (Amblerclassification) among class A beta-lactamases (Matagne A et al., 1998,Biochem J 330: 581-598; Tranier S. et al., 2000, J Biol Chem, 275:28075-28082) Typical amino acid residue at Typical beta-lactamaseposition 276 Len-1, SHV-1, TEM-1, PSE-3/PSE-3, ROB-1 Asn (N) Bacilluscereus 5/B type 1 Bacillus cereus 569/H type 1 Bacillus anthrasis spBacillus licheniformis PenP beta-lactamase Asp (D) Bacillus cereus 569/Htype 3 beta-lactamase Bacillus weihenstephanensis beta-lactamaseBacillus clausii beta.lactamase Staphylococcus aureus PC1 beta-lactamaseSme-1 NmcA IMI-1 beta-lactamases CTX-M enzymes Arg (R) PER-1, VEB-1,CME-1 beta-lactamases Glu (E)

Now, in the present invention, the beta-lactamases comprising an aminoacid sequence having al least 60% sequence identity with SEQ ID NO: 1(Bacillus licheniformis PenP derivate, i.e. P1A derivate) and having ahydrophilic amino acid residue at a position of SEQ ID NO: 1corresponding to position 276 according to Ambler classification, showan extended beta-lactam spectrum as well as improved catalytic effectson beta-lactams.

Before, the role of amino acid substitutions of aspartic acid (D) atposition 276 in resistance to serine beta-lactamase inhibitors or incatalytic properties to various beta-lactams have not been studied amongBacillus spp. beta-lactamases, specifically B. licheniformisbeta-lactamase.

As used herein, the amino acid residue 276 according to Amblerclassification corresponds to amino acid position 243 of SEQ ID NO: 1and amino acid position 249 of SEQ) ID NO: 3.

Typically the beta-lactamases of the present invention have ahydrophilic amino acid at a position corresponding to position 276 ofAmbler classification other than aspartic acid (D). Amino acids areclassified based on the chemical and/or structural properties of theirside chains. The amino acid classification groups include hydrophilicamino acids, which are divided into following groups: polar andpositively charged hydrophilic amino acids; polar and neutral of chargehydrophilic amino acids; polar and negatively charged hydrophilic aminoacids; aromatic, polar and positively charged hydrophilic amino acids.As used herein “hydrophilic amino acid” includes all above-mentionedgroups of hydrophilic amino acids, i.e. refers to polar and positivelycharged hydrophilic amino acids, to polar and neutral of chargehydrophilic amino acids, to polar and negatively charged hydrophilicamino acids and/or to aromatic, polar and positively charged hydrophilicamino acids(http://www.biomed.curtin.edu.au/biochem/tutorials/AAs/AA.html). “Apolar and positively charged hydrophilic amino acid” refers to arginine(R) or lysine (K). “A polar and neutral of charge hydrophilic aminoacid” refers to asparagine (N), glutamine (Q), serine (S) or threonine(T). “A polar and negatively charged hydrophilic amino acid” refers toaspartate (D) or glutamate (E). “An aromatic, polar and positivelycharged hydrophilic amino acid” refers to histidine (H).

In one embodiment of the invention, the hydrophilic amino acid is aneutral or positively charged hydrophilic amino acid selected from thegroup consisting of arginine (R), histidine (H), lysine (K), asparagine(N), glutamine (Q), serine (S) and threonine (T) at a position of Seq IDNo 1 corresponding to position 276 according to Ambler classification.

In a preferred embodiment of the invention, the hydrophilic amino acidof the beta-lactamase at a position of SEQ ID NO: 1 corresponding toposition 276 according to Ambler classification is selected from polarand positively charged hydrophilic amino acids from the group consistingof arginine (R), histidine (H) and lysine (K). Most preferably, theamino acid at the position of SEQ ID NO: 1 corresponding to position 276according to Ambler classification is arginine.

In another preferred embodiment of the invention, the hydrophilic aminoacid is selected from polar and neutral of charge hydrophilic aminoacids from the group consisting of asparagine (N), glutamine (Q), serine(S) and threonine (T). Most preferably, the amino acid at the positionof SEQ ID NO: 1 corresponding to position 276 is asparagine.

In a further preferred embodiment of the invention, the hydrophilicamino acid at the position of SEQ ID NO: 1 corresponding to position 276locates in an alpha helix. An alpha helix is a motif of proteinsecondary structure, resembling a coiled conformation. Alpha helixes mayhave particular significance in DNA binding motifs (e.g.helix-turn-helix, leucine zipper and zinc finger motifs). In a preferredembodiment of the invention, amino acid residue 276 is located at thefinal alpha helix₁₁ (FIG. 1). This alpha helix₁₁ is not conserved amongClass A beta-lactamases.

Specific Features of Class A Beta-lactamases

One specific feature of class A beta-lactamases is a guanidinium groupof Arg278. CTX-M enzymes have Arg278, Arg244 or Arg220, which lies inequivalent positions in the three dimensional structures. Arginine atposition 220 or 244 is shown to be essential for the catalyticproperties of TEM-1 (Leu220 and Arg244) and Streptococus albus Gbeta-lactamase (Arg220 and Asn244). A basic guanidinium group ofarginine 244 or arginine 220 is proposed to contribute the binding ofbeta-lactam or the inactivation chemistry of “suicide” inhibitors suchas clavulanic acid (Matagne et al., 1998, Biochem J. 330:582-598;Perez-Llarena et al., 2008, J Antimicrobial Chemother, 61: 792-797). InB. licheniformis PenP, Arg-244 residue forms a salt bond with aspartaticacid 276 (Herzberg, O. 1991, J Mol Biol. 217: 701-719; Knox, J. R., andP. C. Moews, 1991, J Mol Biol. 220: 435-555).

In a preferred embodiment of the invention, the beta-lactamase furthercomprises at least one amino acid selected from the group consisting ofLeu220 and Arg244 according to Ambler classification, which correspondto Leu189 and Arg212, respectively of SEQ ID NO:1.

Bacillus licheniformis Beta-lactamase (PenP, P1A)

The beta-lactamase of the invention originates from Bacilluslicheniformis 749/C strain. B. licheniformis 749/C beta-lactamase (PenP;penicillin amido-beta-lactamhydrolase, EC3.5.2.6) belongs to a subgroup2a in functional classification of class A beta-lactamases (Bush K. etal., 1995, Antimicrob Agents Chemother 39: 1211-1233). B. licheniformisbeta-lactamase can be considered as a penicillinase, which has highhydrolytic capacity to degrade e.g. penicillin, ampicillin, amoxicillinor piperacillin and it is generally inhibited by active site-directedbeta-lactamase inhibitors such as clavulanic acid, sulbactam ortazobactam (Bush K. et al., 1995, Antimicrob Agents Chemother. 39:1211-1233).

Bacillus licheniformis 749/C beta-lactamase is expressed as apre-protein of 307 amino acid residues. After translocation and removalof its 26 amino acid residues long signal sequence, it becomes amembrane-anchored lipoprotein in which the aminoterminal cysteine (C27)forms a thioether bond with a diacylglyseride. B. licheniformisbeta-lactamase is also found as secreted (extracellular) forms which areproteolytic products of the lipoprotein form (Izui K. et al., 1980,Biochemistry 19: 1882-1886; Matagne A. et al., 1991, Biochem J, 273:503-510). The region of the Bacillus licheniformis 749/C beta-lactamasegene encoding the small, secreted form (small exo form; P1A) of aminoacid residues 43-307 has been chosen as a DNA fragment for tailoring ofhost-vector Bacillus subtilis production system (WO 2008065247).

Function

Beta-lactamases hydrolyse beta-lactam antibiotics comprising abeta-lactam ring such as penicillins, cephalosporins, clavams (oroxapenams), cephamycins and carbapenems. In a preferred embodiment ofthe invention, the beta-lactamase hydrolyses penicillins and/orcephalesporins, “Penicillins” refer to several natural or semisyntheticvariants of penicillin, which is originally derived from Penicillium.Penicillins include but are not limited to amoxicillin, ampicillin,aziocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin,hetacillin, oxacillin, mezlocillin, penicillin G, penicillin V, andpiperacillin.

In cephalosporins, the beta-lactam ring is fused to a six-membereddihydrothiazine ring rather than to the five-membered thiazolidine ringfound in penicillins. Based on their biological activity, cephalosporinsare divided into six generations but some cephaloporins have not beengrouped to a particular generation. In one specific embodiment of theinvention, the beta-lactamase has improved catalytic efficiency oncephalosporins compared to wild type beta-latamases. According topresent invention, Bacillus licheniformis beta-lactamase, in which theaspartic acid (Asp, D) at position 276, numbered in accordance withAmbler classification, is substituted with a hydrophilic amino acidresidue such as an asparagine (N) or arginine (R), exhibits an extendedactivity to beta-lactam antibiotics such as cephalosporins.

In one embodiment of the invention, the cephalosporins are selected fromthe group consisting of cefoperazone, ceftriaxone and cefazoline.

As used herein, catalytic efficiency of beta-lactamases refers to theability to hydrolyse beta-lactam antibiotics. Improved catalyticefficiency can be measured by any conventional in vitro, ex vivo or invivo-methods from any biological sample or a subject.

Methods of Producing and Modifying Beta-lactamases

The beta-lactamase of the invention may be produced by modifying theenzyme with any conventional method of genetic engineering. Methods suchas rational design, random mutagenesis, DNA shuffling (randomrecombination), phage display, whole-genome shuffling, heteroduplex,random chimeragenesis on transient templates assembly of designedoligonucleotides, mutagenic and unidirectional reassembly, exonshuffling, Y-ligation-based block shuffling, nonhomologousrecombination, combination rational design with directed evolution maybe utilized in the production. Furthermore, the mutant enzymes may beobtained by employing site-directed mutagenesis and splicing by overlapextension techniques.

In one embodiment of the invention, a method of modifying abeta-lactamase comprises a step of modifying the beta-lactamasecomprising an amino acid sequence having at least 60% sequence identitywith SEQ ID NO: 1 by replacing an amino acid at a position of SEQ ID NO:1 corresponding to position 276 according to Ambler classification witha hydrophilic amino acid. The hydrophilic amino acid may be anyhydrophilic amino acid, for example selected from the group consistingof arginine (R), histidine (H), lysine (K), asparagine (N), glutamine(C)), serine (S) and threonine (T).

In one embodiment of the invention a non-hydrophilic amino acid isreplaced with a hydrophilic amino acid at a position of SEQ ID NO: 1corresponding to position 276 according to Ambler classification.

The beta-lactamase of the invention can also be produced for example bysynthetic methods e.g. peptide synthesis or by recombinant production ina host cell. In a preferred embodiment of the invention, the enzyme isrecombinant. As used herein, “recombinant” genetic material refers to amaterial, which is typically a combination of genetic material, e.g. DNAstrands of various origin, and it has been produced by combining orinserting the sequences. The polynucleotide of the invention may forexample be inserted under the control of any endogenous or exogenousregulators, such as promoters. Recombinant protein is derived fromrecombinant DNA.

At least one polynucleotide or polynucleotide fragment of interest maybe isolated from a cell or produced synthetically. This polynucleotideor polynucleotide fragment can be transformed to a host cell. A suitablehost cell for producing any peptide of the invention may be anyeukaryotic or prokaryotic cell, preferably bacteria, most preferablyBacillus spp. strain such as Bacillus subtilis, Bacillus licheniformis,Bacillus pumifis, or Bacillus amyloliquefaciens.

As used herein, “transformation” refers to a genetic alteration of acell by foreign genetic material, preferably DNA, resulting inexpression of this genetic material. The foreign genetic material can beintroduced as such or as incorporated into any other genetic materialsuch as vectors, plasmids etc. Any method of genetic engineering or anymolecular cloning methods can be used for transforming a host cell withthe polynucleotide of the invention. There are various methods ofintroducing foreign material into a eukaryotic cell. Materials such aspolymers (e.g. DEAE-dextran or polyethylenimine), liposomes andnanoparticles (e.g. gold) have been used as carriers for transformation.Genetic material can also be introduced into cells by using for exampleviruses or vectors as carriers. Other methods for introducing foreignmaterial into a cell include but are not limited to nucleofection,electroporation, conjucation, transfection, sonoporation, heat shock andmagnetofection.

After a host cell has produced the peptide of the invention inappropriate conditions, the peptide can for example be purified from thecell or a secreted form of the peptide can be recovered e.g. fromculture media. In a preferred embodiment of the invention, thebeta-lactamase is secreted.

Pharmaceutical Composition

The pharmaceutical composition of the invention comprises thebeta-lactamase of the invention. The composition may comprise only onebeta-lactamase or more, such as at least two, three, four etc. differentbeta-lactamases.

The pharmaceutical compositions of the invention may also comprise anyother active ingredients than beta-lactamases of the invention.

The pharmaceutical compositions may be used for example in solid,semisolid or liquid form such as in the form of tablets, pellets,capsules, solutions, emulsions or suspensions. Preferably thecomposition is for oral administration or for enteral applications.

In addition to at least one beta-lactamase of the invention orpolynucleotides or host cells comprising the polynucleotides of theinvention, the pharmaceutical composition may comprise pharmaceuticallyacceptable carrier(s), adjuvant(s), excipient(s), auxiliaryexcipient(s), antiseptic(s), stabilizing agent(s), binding agent(s),filling agent(s), lubricating agent(s), suspending agent(s),plasticizer, colorants, film formers, sugar, alcohols, glidant agentsand diluent agents and/or components normally found in correspondingproducts.

The product or pharmaceutical composition of the invention comprises thebeta-lactamases in an amount sufficient to produce the desired effect.

The products or pharmaceutical compositions may be manufactured by anyconventional processes known in the art. The beta-lactamases may beadded to any pharmaceutical product or mixed with any agents during anypreparation step. The beta-lactamase of the invention may also beproduced for example by expressing the beta-lactamase gene inappropriate conditions in a pharmaceutical product or in the targettissue after the pharmaceutical product has degraded.

In one preferred embodiment of the invention, the beta-lactamase(s) andthe beta-lactam antibiotic are administered together in the form of anenteric coated pellet to a subject. Aqueous-based coating forms appearto be the most favourable materials for coating processes of thehydrophilic P1A protein. The aqueous polymers commonly used to achieveenteric properties, and also usable in the present invention, arepolymethacrylates such as Eudragit®, cellulose based polymers e.g.cellulose ethers e.g. Duodcell®, or cellulose esters, e.g. Aquateric®,or polyvinyl acetate copymers e.g. Opadry®.

Beta-lactamase of the invention or a pharmaceutical composition of theinvention may be administered to a subject simultaneously orsequentially with a beta-lactam antibiotic. In one embodiment of theinvention, the beta-lactamase or the pharmaceutical composition isadministered before a beta-lactam antibiotic, for example 5 to 30minutes before a beta-lactam antibiotic. The beta-lactamase and abeta-lactam antibiotic/antibiotics may be in the same formulation or indifferent formulations.

Adverse Effects of Beta-lactams and Treatments

Adverse effects i.e. adverse drug reactions for the beta-lactamantibiotics may include but are not limited to diarrhea, nausea, rash,urticaria, superinfection, fever, vomiting, erythema, dermatitis,angioedema and pseudo-membranous colitis.

In a preferred embodiment of the invention, the adverse effects to betreated or prevented occur in the gastrointestinal tract (GIT). As usedherein, gastrointestinal tract refers to digestive structures stretchingfrom the mouth to the anus. The gastrointestinal tract comprises themouth, esophagus, stomach, duodenum, jejunum, ileum, small intestine,colon, cecum, rectum and anus.

The beta-lactamase of the invention or the pharmaceutical composition ofthe invention may be administered to a subject orally or directly to thegastrointestinal tract. Drug product(s) of enzyme combinations areintended to inactivate unabsorbed beta-lactam in the GIT or in otherundesired body compartments such as skin or vaginal cavity. Thepharmaceutical composition may be an orally administered drug product, adermatological formulation or a vaginal suppository, and may compriseliquid, immediate, delayed or sustained release dosage formulations.

In one preferred embodiment of the invention, the beta-lactamase(s)is/are administered orally. In another preferred embodiment of theinvention, the beta-lactamase(s) is/are administered directly to thegastro-intestine of a patient.

A treated subject may be a man or an animal such as a pet or productionanimal e.g. dog, cat, cow, pig, chicken or horse. In a preferredembodiment of the invention, the subject is a man.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

Example 1. Construction of D276N and D276R Mutant Enzymes

Bacillus licheniformis beta-lactamase D276N and D276R mutants wereconstructed by splicing-by-overlap extension mutagenesis (SOE) using thepRSH10 plasmid encoding P1A beta-lactamase as a template for the initialPCR reactions according to previously published procedures (Horton R. M.et al., 1989, Gene 77:61-68). Primers were designed to provide twodifferent PCR products with a region of common sequence. Fragments werethen fused in a subsequent PCR amplification by aid of overlappingregions. The desired mutations were achieved by using mutagenic primersin initial PCR.

For the D276N mutant, mutation was made at the desired position in wildtype gene, converting a GAT codon to a AAT codon. The primers utilizedin the first PCR amplifications are presented in Table 2. The size ofamplified fragments in the first PCR was 800 nt and 220 nt which have a21 nt long overlapping region.

TABLE 2 Oligonucleotide PCR primers. Complementary regions are shadedand mutated codons are expressed as bold. Forward-1 and reverse-1primers were used in amplification of fused fragment in the second PCR.Size of PCR fragment (nt) Oligonucleotide primers 800Forward-1: 5′-CGA TTG TTT GAG AAA AGA-3′ (SEQ ID NO: 4)Reverse-D276N: 5′-AAT AAG TTT ATT ATC ATA CTT GCG GTC CT-3′(SEG ID NO: 5)Reverse-D276R: 5′-AAT AAG TTT GCG ATC ATA CTT GGC GTC CT-3′(SEQ 10 NO: 6) 220Forward-D276N: 5′-AAG TAT GAT AAT AAA CTT ATT GCA GAG G-3′(SEQ ID NO: 7)Forward-D276R: 5′-AAG TAT GAT CGC AAA CTT ATT GCA GAG G-3′(SEQ ID NO: 8) Reverse-1: 5-GTA TTT GTC ACA CCT GAT G-3′ (SEQ ID NO: 9)

In the second PCR reaction (SOE reaction), the two overlapping fragmentswere fused together in a subsequent extension reaction. The inclusion ofoutside primers (Forward-1 and Reverse-1) in the extension reactionamplifies the fused product by PCR. The purified SOE product wasdigested with HindIII restriction enzyme and ligated to HindIII cleavedpKTH141 secretion vector as described in WO 2008/065247.

Competent cells of Bacillus subtilis RS303 were transformed with aligation mixture. Positive dories on Luria-kanamycin plates werescreened by suspending bacterial mass of a single colony into nitrocefinsolution. Positive clones effectively hydrolyzed nitrocefin turning thecolour of nitrocefin solution from yellow to red. Hybrid plasmid waspurified from cells of a single clone. The correct sequence of PCRgenerated region was verified by DNA sequencing.

For the D276R mutant, mutation was made at the desired position byconverting a GAT codon to a CGC codon. Construction of D276R mutantstrain was performed similar to that of D276N mutant exceptreverse-D276R- and forward-D276R-primers were used in the initial PCR(see Table 2).

Example 2. Nucleotide Sequence of D276N Mutant Beta-lactamase Gene(penP)

The expression construct was isolated from a positive clone and theinsert was subjected to DNA sequencing. The complete nucleotide sequenceand deduced amino acid sequences of D276N mutant beta-lactamase generevealed that a substitution of Asp for Asn has occurred correctly atthe desired codon (FIG. 2). Furthermore, the DNA sequence of D276Nmutant beta-lactamase gene revealed in frame fusion between nucleotidesequence encoding a 31 amino acid long signal sequence of Bacillusamyloliquefaciens alpha amylase, the HindIII cloning site and thecomplete sequence of D276N mutant gene. The signal peptidase ispredicted to cut the peptide bond between alanine (A) at position of −1and glutamine (Q) at position of +1. The mature D276N beta-lactamasepossesses an NH₂-terminal extension of a NH₂-QAS-tripeptide derived fromthe HindIII cloning site in the expression construct. Hence, based onthe deduced amino acid sequence the mature D276N mutant beta-lactamaseis comprised of 268 amino acid residues.

Example 3. Nucleotide Sequence of D276R Mutant Beta-lactamase Gene(penP)

To confirm the desired substitution of aspartic acid to arginine atposition 276 (Ambler classification) in the Bacillus licheniformisbeta-lactamase gene, the expression construct was isolated from apositive clone and the nucleotide sequence of the insert was sequencedsimilar to example 2. According to the obtained nucleotide sequence, thededuced amino acid sequence contains the desired D276R substitution andthe mature D276R mutant enzyme is comprised of 268 amino acid residues(FIG. 3).

Example 4. Biochemical Analysis of D276N Mutant Beta-lactamase (P3A)

The purity of the enzyme preparate was estimated to more than 95percentages by SDS-PAGE analysis (data not shown).

Kinetic parameters of the wild type (P1A) and D276N (P3A) mutant B.licheniformis beta-lactamases were determined for hydrolysis of varioustypes of beta-lactams and are summarized in Table 3. Enzymatic reactionswere performed in 20 mM phosphate buffer (pH 7) at 30° C. by usingappropriate enzyme concentration and various concentrations ofpenicillin or cephalosporin substrates. The k_(cat) and K_(m) valueswere obtained with the aid of the Hanes linearization method. The mainresults are described below.

(i) Penicillins

The effect of the D276N substitution on the hydrolysis of penicillins(ampicillin amoxicillin or piperacillin) was not drastic with enzymaticefficiencies of 51-80 percentages of those of the wild type enzyme.Consequently, k_(cat)/K_(m) values of D276N mutant enzyme forpenicillins were reduced as a maximum of two folds or less.

(ii) Cephalosporins

As expected, related to penicillins, the wild type beta-lactamase hadpoor enzymatic efficiencies for various cephalosporins including thefirst (cafazoline), the second (cefuroxime), and the third (ceftriaxone,cefotaxime, ceftadizime, cefoperazone, and cefepime) generationcephalosporins (Table 1). Surprisingly, the enzymatic efficiencies ofD276N mutant enzyme for certain cephalosporins, preferably forcefoperazone and more preferably for ceftriaxone, were essentiallyimproved compared to those obtained with wild type enzymes. The K_(m)constants for ceftriaxone and cefoperazone were decreased andconcomitantly the turnover numbers (k_(cat)) for ceftriaxone andcefoperazone were increased compared to those of the wild type enzyme(P1A). Thus the aspartic acid—asparagine substitution at position 276 ofBacillus licheniformis beta-lactamase contributes the extension ofbeta-lactam substrate profile in Bacillus licheniformis beta-lactamase.

TABLE 3 Kinetic parameters for hydrolysis of beta-lactam substrates bywild type (P1A) and D276N mutant enzymes of Bacillus licheniformisbeta-lactamases. Wild type beta- Relative lactamase (P1A) D276N mutantcatalytic K_(m) k_(cat) k_(cat)/K_(m) K_(m) k_(cat) k_(cat)/K_(m)efficacies Beta-lactam (μM) (s⁻¹) (μM⁻¹ s⁻¹) (μM) (s⁻¹) (μM⁻¹ s⁻¹) (%)⁽¹Ampicillin 157 3369 21.45 161 2160 13.42 63 Piperacillin 49 939 19.16 53816 15.40 80 Amoxicillin 119 2956 24.84 219 2789 12.74 51 Ceftriaxone400 0.045 0.00013 38 83 2.18 1676923 Cefotaxime 363 246 0.67 213 35 0.1725 Ceftadizime 0 0 0 1505 2.74 0.0018 Cefepime 0 0 0 1357 133 0.1Cefazoline 22 93 4.22 37 192 5.19 123 Cefoperazone 7 10 1.43 2 17 8.2573 Cefuroxime 107 233 2.18 277 35 0.13 6 ⁽¹Relative catalyticefficiency (k_(cat)/K_(m)) of D276N compared to that of the wild typeenzyme (P1A).

Example 5. Biochemical Characterization of D276R Mutant Enzyme

D276 mutant enzyme was constructed to evaluate whether Asp-276 toleratessubstitutions and assesses the contribution of D276R substitution to theextension of beta-lactamase activity observed in D276N enzyme.

Crude enzyme samples of D276R and D276N obtained from culturesupernatants were employed as test materials. The purity and quantity ofenzyme samples were estimated by performing SDS-PAGE-analysis.Hydrolysis rate of D276R and D276N mutant enzymes for variousbeta-lactams were performed by determining V_(max) values. Obtainedresults are presented as relative activities (%) compared to those ofD276N enzyme in Table 4.

In general, catalytic efficiencies of D276R beta-lactamase for bothpenicillins and cephalosporins are comparable to those of D276N enzyme.In comparison with 0276N enzyme, D276R enzyme has reduced activity toceftriaxone and improved activity to cefoperazone. This study showedthat the extended spectrum of beta-lactams can be achieved bysubstituting a hydrophilic amino acid residue such as arginine orasparagine for aspartic acid at position 276 in the Bacilluslicheniformis beta-lactamase. It also indicates that a desired enzymemodification can be achieved by substituting another hydrophilic aminoacid residue such as glutamine (Q), lysine (K), serine (S) or threonine(T) for aspartic acid at position 276.

TABLE 4 Relative activities (%) of D276R mutant enzyme compared to thoseof D276N mutant enzyme Beta-lactam Relative activities Ampicillin 82Piperacillin 84 Amoxicillin 71 Ceftriaxone 50 Cefotaxime 105 Ceftadizime— Cefepime 74 Cefazoline 84 Cefoperazone 232 Cefuroxime 99

Example 6. In Vivo Study of D276N Beta-lactamase

The capability of Bacillus licheniformis D276N mutant beta-lactamaseenzyme to inactivate ceftriaxone (CRO) which has been excreted into thegastrointestinal tract during parenteral therapy was investigated in adog model. Laboratory beagles of the study have a nipple valvesurgically inserted in jejunum approximately 170 cm distal to pylorusenabling collection of samples for the analysis. The intestinal surgerydid not alter the intestinal motility. Five beagle dogs were utilized ineach experiment.

The study was performed as two sequential treatments: In the firsttreatment (control experiment without beta-lactamase therapy), a singledose of ceftriaxone (30 mg of ceftriaxone (CRO) per kg of body weightwhich corresponds to about 1 gram dose of CRO in humans) wasadministered intravenously 20 minutes after the first feeding of thedogs. Jejunal samples were collected at various time points during tenhours. The dogs were fed again five hours and forty minutes after theceftriaxone administration in order to induce the biliary excretion ofceftriaxone accumulated in gallbladder.

Jejunal chyme samples were immediately freezed and stored at −20° C. toawait the analysis. Chyme samples were pretreated with perchloric-citricacid in order to precipitate interfering substances. The precipitateswere removed by centrifugation. A reverse-phase high-pressurechromatography method with UV detection was used for the quantificationof ceftriaxone in supernatants.

In the second treatment, D276N mutant beta-lactamase was given asenteric coated pellets filled in hard gelatine capsules 10 minutes priorto ceftriaxone injection. Enteric coating dosage forms are common amongoral products in pharmaceutical industry. Enteric coating drug productsare designed to bypass stomach as an intact form and to release thecontents of the dosage form in small intestine. The reasons for applyingenteric solid formulations are to protect the drug substance from thedestructive action of the enzymes or low pH environment of stomach or toprevent drug substance-induced irritation of gastric mucosa, nausea orbleeding or to deliver drug substance in undiluted form at a target sitein small intestine. Based on these criteria, enteric coated drugproducts can be regarded as a type of delayed action dosage forms.Polymethacrylic acid copolymer Eudragit® L 30 D-55 was employed in orderto achieve a pH dependent enteric-coated dosage form. A single dose ofenteric coated pellets containing about 0.44 mg of active D276Nbeta-lactamase per kg of body weight was used in the second treatment.

Obtained results from both treatments are presented in FIG. 4. Treatment1 showed that high concentrations of ceftriaxone were excreted into thesmall intestinal tract during the parenteral ceftriaxone therapy. Thehighest jejuna concentration (about 1500 micrograms per gram of jejunalchyme) was found 60 minutes after the ceftriaxone injection. Theincreased jejunal ceftriaxone levels were observed after the secondfeeding of the dogs (at time point 340 minutes) which indicates foodstimulated, ceftriaxone containing bile excretion accumulation ingallbladder.

Treatment 2 showed that orally administered D276N mutant beta-lactamaseis capable to reduce jejunal ceftriaxone levels near to the limit ofquantification (10 micrograms of ceftriaxone per microgram of jejunalchyme). This finding shows that D276N mutant beta-lactamase is a potentdrug substance candidate for reducing the side effects related to a useof parenteral ceftriaxcne. Moreover, based on high activities topenicillins such as ampicillin, amoxicillin and piperacillin, D276N orD276R mutant enzymes can be used as an alternative drug substance inbeta-lactamase therapy described in WO 2008065247.

The invention claimed is:
 1. A method for reducing the likelihood of a Clostridium difficile infection in a subject in need thereof, comprising administering a beta-lactamase to the subject wherein: the subject is receiving a beta-lactam antibiotic and the Clostridium difficile infection is caused by unabsorbed beta-lactam antibiotic in the intestinal tract, the beta-lactam antibiotic being a substrate of the beta-lactamase, and the beta-lactamase comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 1 and an asparagine (N) or arginine (R) residue at a position corresponding to position 276 according to Ambler classification.
 2. The method of claim 1, wherein the beta-lactamase hydrolyses a penicillin and a cephalosporin.
 3. The method of claim 2, wherein the cephalosporin is selected from cefoperazone and ceftriaxone.
 4. The method of claim 1, wherein the beta-lactamase is administered simultaneously with or sequentially to the beta-lactam antibiotic.
 5. The method of claim 4, wherein the beta-lactamase is administered before the beta-lactam antibiotic.
 6. The method of claim 1, wherein the beta-lactamase is administered orally.
 7. The method of claim 1, wherein the beta-lactam antibiotic is administered by intravenous injection.
 8. The method of claim 1, wherein the method maintains an ecological balance of normal intestinal microbiota.
 9. The method of claim 1, wherein the beta-lactamase comprises the amino acid sequence of SEQ ID NO: 1 with an asparagine (N) residue at position 276 according to Ambler classification.
 10. The method of claim 1, wherein the beta-lactamase comprises the amino acid sequence of SEQ ID NO: 1 with an arginine (R) residue at position 276 according to Ambler classification. 